Interactions of TOM1L1 with the Multivesicular Body Sorting Machinery*

Tom1L1 (Tom1-like1) and related proteins Tom1 (Tar-get of Myb1) and Tom1L2 (Tom1-like2) constitute a new protein family characterized by the presence of a VHS (Vps27p/Hrs/Stam) domain in the N-terminal portion fol- lowed by a GAT (GGA and Tom) domain. Recently it was demonstrated that the GAT domain of both Tom1 and Tom1L1 binds ubiquitin, suggesting that these proteins might participate in the sorting of ubiquitinated proteins into multivesicular bodies (MVBs). Here we report a novel interaction between Tom1L1 and members of the MVB sorting machinery. Specifically, we found that the VHS domain of Tom1L1 interacts with Hrs (Hepato- cyte growth factor-regulated tyrosine kinase substrate), whereas a PTAP motif, located between the VHS and GAT domain of Tom1L1, is responsible for binding to TSG101 (tumor susceptibility gene 101). Myc epitope- tagged Tom1L1 showed a cytosolic distribution but was recruited to endosomes following Hrs expression. In ad-dition, Tom1L1 possesses several tyrosine motifs at the C-terminal region that mediate interactions with members of the Src family kinases and other signaling proteins such as Grb2 and p85. We showed that a fraction of Fyn kinase localizes at endosomes and that this distribution becomes more evident after epidermal growth factor internalization. Moreover, expression of a constitutive active form of Fyn also promoted the recruitment of Tom1L1 to enlarged endosomes. Taken together, we propose that Tom1L1

Regulated degradation of cell surface proteins, particularly receptors involved in signaling pathways, is essential for the control of many biological processes, including cell proliferation, survival, migration, and differentiation (1,2). Internalized membrane-protein cargo can be delivered either to recycling vesicles for transport back to the cell surface or to late endosomes for transfer to lysosomes and degradation. Multivesicular bodies (MVBs) 1 are the compartments where this sort-ing event takes place (3). Thus, proteins to be degraded are included into small vesicles that invaginate into the lumen of the endosome. Subsequent fusion of MVBs with lysosomes releases these vesicles into the lysosome lumen, where they are degraded by lysosomal hydrolases. In contrast, proteins that remain in the limiting membrane of MVBs are recycled to the trans-Golgi network (TGN) or plasma membrane. In the last few years, a number of genetic and biochemical studies have allowed the characterization of the molecular machinery implicated in the formation of MVBs. Hrs appears to be the first protein to be recruited to endosomes (4 -6) through an interaction of its FIVE domain with phosphatidylinositol 3-phosphate (7), a lipid highly enriched in endosomal membranes (8). This is followed by the consecutive recruitment of three multisubunit complexes termed ESCRT-I (9), ESCRT-II (10), and ESCRT-III (11). Finally, the AAA-type ATPase Vps4 binds ESCRT-III and, following MVB vesicle formation, promotes the dissociation of the ESCRT protein complexes from endosomes for additional rounds of protein sorting (12). Numerous studies have shown that mono-ubiquitination of cargo can serve as a signal for recognition by the vacuolar sorting machinery (13)(14)(15). Several components of the MVB sorting machinery have specific domains that allow them to interact with ubiquitinated cargo; this is the case with the ubiquitin-interacting motif of Hrs (16) and the UEV domain of TSG101 (17).
Recently, the GAT domain present in the GGA (Golgi-localizing, ␥-adaptin ear domain homology, ADP-ribosylation factor (Arf)-binding protein) and Tom families of proteins has been shown to bind ubiquitin (18 -21). In addition, a role for these proteins in the sorting into MVBs has also been suggested. GGA proteins are monomeric adaptors that are recruited to the TGN by the Arf-1 GTPase (22)(23)(24)(25) and mediate the transport of lysosomal enzymes. They consist of four distinct segments as follows: a VHS domain that binds an acidic di-leucine sorting signal found in the mannose 6-phosphate receptor and other proteins known to cycle between TGN and endosomes (26,27); a GAT (GGA and Tom) domain that interacts with the GTPbound form of Arf (22)(23)28); a hinge region that recruits clathrin (27,28); and a similar to the ␥-adaptin ear domain that exhibits sequence similarity to the ear region of ␥-adaptin and recruits a number of accessory proteins (24, 29 -31). Most interestingly, we have recently described (18) that GGAs are present not only at the TGN but also at endosomes. Knockdown of GGA3 by RNA interference had a very clear effect on the MVB pathway and resulted in the accumulation of enlarged endosomes and a block in transport of EGF receptor molecules to lysosomes. Moreover, the GAT domain of GGAs has the ability to interact with both ubiquitin and the ESCRT-I component TSG101 (18,32), thus indicating a potential role in endosomal sorting.
In humans, the Tom family comprises three members termed Tom1, Tom1L1, and Tom1L2. Initially, Tom1 was identified as a protein whose expression is induced by v-Myb (33) and more recently was suggested to act as a negative regulator of the signaling pathways induced by IL-1␤ and TNF-␣ (34). All Toms have a structure similar to GGAs including a VHS domain at the N-terminal region followed by a GAT domain. However, despite the sequence homology, the VHS domain of Tom1 and Tom1L1 does not contain the specific pocket implicated in the binding to acidic di-leucine sorting signal sequences (35,36), whereas the GAT domain does not conserve a helical extension necessary for the association with Arf (37)(38)(39). Therefore, the recent finding that the GAT domain of GGAs and Toms can interact with ubiquitin is of special interest, as it represents the first functional characteristic common to both families.
To determine whether Toms also participate in the regulation of MVB formation, we decided to address whether these proteins can interact with components of the MVB sorting machinery. In the present work, we characterize the interaction of Tom1L1 with TSG101 and Hrs, as well as the recruitment of Tom1L1 to endosomes after Hrs or active Fyn overexpression. We feel the presence of motifs in the Tom1L1 C-terminal region that mediates interactions with signaling proteins, such as the tyrosine kinase Fyn and Grb-2, and the regulatory subunit of phosphoinositide 3-kinase, p85 (40), makes this protein an excellent candidate to connect signaling and degradative pathways.

Plasmids-
The complete open reading frame of human Tom1L1 (GenBank TM accession number AJ010071) was PCR-amplified from a human pancreas cDNA library using specific primers and was cloned into pcDNA3.1/V5-His Topo vector (Invitrogen). To generate Tom1L1myc, the 3Ј primer included an additional sequence to place a Myc epitope at the C terminus of the protein. Constructs encoding Tom1L1-VHS (residues 1-154), Tom1L1-GAT (residues 200 -284), Tom1L1-VH-SGAT (residues 1-284), and Tom1L1 (residues 285-476) were obtained by PCR amplification and were cloned into the EcoRI-SalI sites of pGAD424 (Clontech). Mutation of 179 PTAP motif to alanines was introduced using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Fyn tyrosine kinase was amplified from a human heart cDNA library and cloned into pEGF-N3 via SalI-SacII. To generate constitutive active Fyn, tyrosine 531 was substituted by phenylalanine by introducing a punctual mutation into the 3Ј primer. Plasmids encoding the following proteins were gifts of the investigators as indicated in parentheses: TSG101 (S. Cohen; Stanford University), GFP-hVps4-EQ (P. Woodman; University of Manchester, UK), and GFP-Hrs (Sylvie Urbe; University of Liverpool, UK).
Antibodies and Reagents-Mouse monoclonal anti-Myc antibody (9E10) was obtained from Covance (Princeton, NJ). A polyclonal antibody to Hrs was the kind gift of Sylvie Urbe (University of Liverpool,) (41,42). Epidermal growth factor complexed to Alexa 555 was supplied by Molecular Probes (Eugene, OR).
Transfection and Immunofluorescence Microscopy-Transfection of HeLa cells was performed by using FuGENE 6 (Roche Applied Science). For immunofluorescence, transfected HeLa cells were grown on coverslips, fixed in methanol/acetone (1:1 v/v) for 10 min at Ϫ20°C, and subsequently air-dried. Incubation with primary antibodies diluted in phosphate-buffered saline (PBS), 0.1% (w/v) saponin, 0.1% bovine serum albumin was carried out for 1 h at room temperature. Unbound antibodies were removed by rinsing with phosphate-buffered saline for 5 min, and cells were subsequently incubated with a secondary antibody (Cy3-conjugated donkey anti-rabbit or anti-mouse Ig) diluted in PBS, 0.1% (w/v) saponin, 0.1% bovine serum albumin, for 30 -60 min at room temperature. After a final rinse with PBS, coverslips were mounted onto glass slides with Fluoromount G (Southern Biotechnology Associates, Birmingham, AL). Fluorescence images were acquired on an LSM 510 confocal microscope (Carl Zeiss, Thornwood, NY). For EGF internalization assays, cells were starved for 6 h in serum-free medium and then stimulated with EGF complexed to Alexa 555 (Molecular Probes, Eugene, OR) at 200 ng/ml for various times.
Yeast Two-hybrid Assays-The Saccharomyces cerevisiae strain HF7c was transformed by the lithium acetate procedure as described in the instructions for the MATCHMAKER two-hybrid kit (Clontech). For colony growth assays, two colonies of each HF7c transformant were resuspended in water to 0.1 A 600 /ml, and 5 l was then applied to plates lacking leucine and tryptophan or leucine, tryptophan, and histidine and allowed to grow at 30°C for 3-4 days.

Tom1L1
Interacts with TSG101-To access the ability of Tom1L1 to bind ubiquitinated proteins and to determine whether this may indicate a role for this protein in the MVB pathway, we analyzed whether Tom1L1 can interact with components of the MVB sorting machinery. Yeast two-hybrid experiments revealed that an N-terminal fragment of Tom1L1 that includes both the VHS and GAT domains interacts with the full-length version of TSG101, a component of the ESCRT-I complex (Fig. 1, A and B). Next, we examined which domain of TSG101 was responsible for the binding to Tom1L1. As shown in Fig. 1C, the ubiquitin E2 variant (UEV) domain of TSG101, which comprises the first 150 amino acids of the protein, bound the VHS-GAT region of Tom1L1 with the same efficiency as full-length TSG101. In contrast, a fragment of TSG101 that included the C-terminal portion of the protein (from amino acid 150 to 390) failed to interact with Tom1L1. The UEV domain of TSG101 is related to the E2 enzymes that ligate ubiquitin to substrate proteins but is enzymatically inactive because it lacks the catalytically important cysteine residue (43,44). Previously it was described that the UEV domain of TSG101 binds both ubiquitin and the PTAP/PSAP tetrapeptide motif located within the late domain of some viral proteins and Hrs (5,(45)(46). Most importantly, the analysis of the Tom1L1 sequence revealed the presence of a PTAP motif located between the VHS and GAT domain. Moreover, the mutation of the 179 PTAP motif to alanines abolished the interaction with both TSG101 full-length and TSG101-UEV domain (Fig. 1, D and E). Thus, we characterized a novel interaction between the UEV domain of TSG101 and a PTAP motif located between the VHS and GAT domain of Tom1L1.
Interaction between the VHS Domain of Tom1L1 and Hrs-To determine whether Tom1L1 binds other components of the MVB sorting machinery, we performed additional two-hybrid experiments. Specifically, we analyzed whether Tom1L1 can interact with Hrs, a key protein in the regulation of protein degradation ( Fig. 2A). Fig. 2B reveals an interaction between the VHS domain of Tom1L1 and full-length Hrs. In agreement with these data, a construct containing both the Tom1L1 VHS and GAT domains also bound Hrs, whereas the Tom1L1 GAT domains alone and the C-terminal portion of the protein (Tom1L1-(285-476)) demonstrated no apparent binding (Fig.  2B). The region of Hrs responsible for the binding to Tom1L1 comprises the residues located between position 450 and 775 (Fig. 2C). This region includes a coiled-coil domain that has been implicated previously in the binding to STAM (47) and TSG101 (48). Most interestingly, the ability of Tom1L1 VHS domain to interact with Hrs could be extended to other VHS domains. For example, the VHS domains of Tom1 and GGA family members (GGA1, GGA2, and GGA3) all bound Hrs with varying efficiency (Fig. 2D).
Hrs Recruits Tom1L1 to Endosomes-To examine the physiological relevance of the observed interaction between Tom1L1 and members of the MVB sorting machinery, we examined whether Tom1L1 localized to endosomes. Transient expression of a Myc epitope-tagged Tom1L1 in HeLa cells revealed a cytosolic distribution for Tom1L1 (Fig. 3A). However, this is not unusual for members of the Tom family. Previous studies (49) have reported that Tom1 localizes to the cytosol, but it can be recruited to endosomes following the overexpression of the endosomal protein Toll-interacting protein (Tollip). Analogously, we found that the overexpression of GFP-Hrs, which localizes to endosomes (50), was able to redistribute Tom1L1 from the cytosol to endosomes. Fig. 3 (B-D) shows that all the GFP-Hrs-positive structures also contain Tom1L1-myc. In addition, expression of a dominant-negative ATPase-deficient mutant of human Vps4 (hVps4-EQ), which stabilizes association of TSG101 and other components of the ubiquitin sorting machinery with aberrantly enlarged MVB precursor endosomes (51), resulted in the accumulation of Tom1L1-myc on those endosomes (Fig. 3, E, F, and G). In contrast with the complete colocalization observed between GFP-Hrs and Tom1L1-myc, it is interesting to note that the colocalization between GFP-hVps4-EQ and Tom1L1-myc is only partial (Fig.  3J, inset). Although both proteins are distributed on the enlarged endosomes, this could reflect that there is not a close contact between them. On the basis of these observations, we conclude that the interactions involving Tom1L1 and components of the endosomal sorting machinery observed by twohybrid experiments are also occurring in cells.
Localization of Fyn on Endosomes after EGF Internalization-It has been described previously that Srcasm (Src activating and signaling molecule), the mouse homolog of Tom1L1, is capable of interacting with Fyn, Grb2, and the regulatory subunit of phosphoinositide 3-kinase, p85, in a phosphorylationdependent manner through several SH2 and SH3 domains located in the C-terminal portion of Srcasm (40). The motifs responsible for these interactions are conserved between mouse and human, indicating that Tom1L1 is probably also able to bind the same proteins. In fact, analogous to what happens with Srcasm, we have observed that the expression of a constitutive active Fyn promotes the tyrosine phosphorylation of Tom1L1 (data not shown). The ability of Tom1L1 to interact with both components of the MVB sorting machinery and proteins implicated in signal transduction prompted us to consider that Tom1L1 might be acting to connect degradative and signaling pathways. If that is the case, we should be able to detect at least a small proportion of these signaling proteins at endosomes. To address this question, we analyzed the distribution of Fyn tyrosine kinase fused to green fluorescent protein (Fyn-GFP). As shown in Fig. 4A, the transient expression of Fyn-GFP in HeLa cells showed that although most of the protein localized at the plasma membrane, some intracellular vesicles containing Fyn-GFP were also observed. To identify the nature of these vesicles, EGF internalization was assessed over time. Most interestingly, endocytosed EGF reached Fyn-GFP-positive structures after 5 min of internalization from the plasma membrane (Fig. 4, C-E). The colocalization between both proteins became more evident after 10 (Fig. 4, F-H) or 20 min of internalization (Fig. 4, I-K). Moreover, the amount of Fyn-GFP localized at endosomes also increased as EGF endocytosis proceeded. These results are in agreement with previous reports (52)(53)(54) that have proposed a participation of the Src family kinases (SFKs), which includes Fyn, in signaling at endosomes.
A Constitutive Active Fyn Kinase Promotes the Recruitment of Tom1L1 to Enlarged Endosomes-We also examined the intracellular distribution of a constitutive active version of Fyn (Fyn-Y531F-GFP). Most interestingly, the vesicular localization of Fyn-Y531F-GFP was much more apparent than that observed for Fyn wild type (compare Fig. 4, A and B). The observed vesicles likely correspond to endosomes, as they colocalize with the endosomal protein Hrs (Fig. 5, A-C). Further- Yeast transformants bearing the combination of constructs indicated were spotted onto media lacking leucine and tryptophan with or without histidine (ϩHis and ϪHis, respectively).

FIG. 3. Recruitment of Tom1L1 to endosomes after Hrs overexpression.
HeLa cells were transfected with plasmids encoding Tom1L1-myc alone (A) or in combination with GFP-Hrs (B-D) or GFP-hVps4-EQ (E, F, and G). Transfected cells were fixed, permeabilized, and immunostained with a monoclonal antibody to Myc followed by Cy3-conjugated donkey anti-mouse IgG. Cells were examined by confocal fluorescence microscopy. B and E, GFP fluorescence (green); A, C, and F, Tom1L1-myc (red). Merging the images in the red and green channels generated the third picture on each row (D and G); yellow indicates overlapping localization. The insets show 2-fold magnification of the structures pointed by the arrows. Scale bar represents 10 m.

TOM1L1 Interacts with Hrs and Tsg101 9261
more, the overexpression of Fyn-Y531F-GFP induced an enlargement of endosomal structures similar to the one observed after accumulation of ubiquitinated proteins or defects on the MVB sorting machinery (Fig. 5, A-C). This effect could be due to the hyper-phosphorylation of Hrs (55) or other endosomal proteins. In addition, the overexpression of Fyn-Y531F-GFP also resulted in recruitment of Tom1L1 to enlarged endosomes (Fig. 5, D-F, arrows). Taken together, the results suggest that Fyn, Hrs, and Tom1L1 can colocalize to the same endosomal structures.

DISCUSSION
Three different studies (18 -20) have recently suggested that the GGAs might play a role in the sorting of ubiquitinated cargo at endosomes. The high degree of structural similarity between the GGAs and Tom1L1, all which contain VHS and GAT domains, together with the fact that the GAT domain of Tom1L1 interacts with ubiquitin, prompted us to analyze whether Tom1L1 also participates in the delivery of ubiquitinated cargo into MVBs. Here we report that Tom1L1 interacts with Hrs and TSG101, two components of the MVB sorting machinery, and that the overexpression of GFP-Hrs or GFP-hVps4-EQ promotes the recruitment of Tom1L1 from the cytosol to the endosomal membranes. The interaction of Tom1L1 and TSG101 was not completely surprising. It has been described recently that the C-terminal portion of the GGAs GAT domain is a three helix bundle that mediates interactions with different effectors, including Rabaptin-5, ubiquitin, and TSG101 (18,30,32). The fact that the GAT domain of Tom1 and Tom1L1 is able to interact with ubiquitin led us to hypothesize that it might also bind TSG101. As predicted, we found an interaction between Tom1L1 and TSG101. However, in contrast to the GGAs, this interaction is not mediated by the GAT domain but by a PTAP motif located in a 56-amino acid segment between the Tom1L1 VHS and GAT domain. In support of this finding, a residue found to be critical for the binding of GGAs to TGS101 (GGA1-Leu-277) is not conserved in the Tom1L1 GAT region. In addition, the segment that contains the PTAP domain is predicted to adopt a coiled-coil conformation when analyzed using the COILS secondary structure prediction program (56) (www.ch.embnet.org/software/COILS_ form.html). This is consistent with its presumed participation in protein-protein interactions. Our finding here that the VHS domain of Tom1L1 interacts with Hrs is also of particular significance. Most importantly, the observed VHS-Hrs interaction extends beyond just Tom1L1, as we also demonstrate Hrs binding to the VHS domains of Tom1 and the GGAs.
Based on the results presented here, we propose that there are not only structural similarities but also functional similarities between GGAs and Toms. Furthermore, these features might reflect a common role in endosomal sorting. Similarities include interactions with Hrs and ubiquitin through their VHS and GAT domains, respectively, and interaction with TSG101. In addition, GGAs and Toms undergo mono-ubiquitination (20,21), they can localize at endosomes (18,49), and they are capable of binding to clathrin heavy chain through typical clathrin-binding motifs (21,28).
In contrast to the GGAs and other members of the Tom family, Tom1L1s possess some very specific characteristics that suggest this protein might participate in signaling. Srcasm, the Tom1L1 murine ortholog, contains motifs predicted to interact with the SH2 and SH3 domains of SFKs, as well as the SH2 domains of Grb2 and p85 phosphoinositide 3-kinase. In addition, Srcasm can serve as a Fyn substrate and activate Fyn kinase activity. Recent lines of evidence have suggested that signaling can also occur at endosomes (52,53) and that regulation of receptor trafficking might modulate the specificity and the duration of the signal transduction process. Furthermore, EGF receptor complexes remain active (57) and associated with effectors, such as Grb2 and Ras, on endosomal membranes (54,58). p85/p110 phosphoinositide 3-kinase is another molecule that is recruited to endosomes after growth factor stimulation (59,60). In line with this these results, we have found that a subpopulation of Fyn-GFP localizes with internalized EGF on endosomes, and this colocalization is more evident after 20 min of internalization. Consistently, Src, another member of the SFKs, also localizes partially to endosomes (55,61,62). Therefore, the three proteins that have been described to interact with the C-terminal region of Tom1L1, namely Fyn, Grb2, and p85, are known to be translocated to endosomes after EGF receptor internalization. In addition, active Fyn-GFP promoted the recruitment of Tom1L1 itself from cytosol to endosomal membranes, indicating that the capacity of Tom1L1 to bind signaling molecules may be related to its proposed role in sorting into MVBs and protein degradation.
These results raise the interesting question of how Tom1L1 might connect signaling and protein degradation at endosomes. One possibility is that Tom1L1 could act as a bridge helping to bring in contact activated signaling complexes with the protein degradation machinery. Alternatively, Tom1L1 could facilitate the SFKs-dependent phosphorylation of some of the components of the MVB sorting machinery (55) regulating their association with endosomal membranes.
Recently, it has been described that Tom1, another member of the Tom family, can act as a common suppressor in both IL-1␤-and TNF-␣-dependent pathways. Most importantly, the VHS domain of Tom1 is indispensable for this function (34).
Here we described an interaction between the VHS domain of Tom1 and Hrs, suggesting that the inhibitory effect on IL-1␤ and TNF-␣ signaling could be related to protein degradation. Further studies will determine whether Toms have a general role in the sorting of ubiquitinated protein or in regulating trafficking and/or activity of specific receptors.